Luteolin target HSPB1 regulates endothelial cell ferroptosis to protect against radiation vascular injury

Abstract

Vascular endothelial damage due to ionizing radiation is the main pathological process of radiation injury and the main cause of damage to various organs in nuclear accidents. Ferroptosis plays an important role in ionizing radiation-induced cell death. We have previously reported that luteolin is highly resistant to ferroptosis. In the present study, body weight, microvessel count, H&E, and Masson staining results showed that luteolin rescued radial vascular injury in vivo. Cell Counting Kit 8 (CCK8), Giemsa staining clarified the anti-ferroptosis ability of luteolin with low toxicity. Malondialdehyde (MDA), superoxide dismutase (SOD), NADP+/NADPH, Fe2+ staining, dihydroethidium (DHE) and MitoTracker assays for ferroptosis-related metrics, we found that luteolin enhances human umbilical vein endothelial cells (HUVECs) antioxidant damage capacity. Drug affinity responsive target stability (DARTS), surface plasmon resonance (SPR), computer simulated docking and western blot showed that heat shock protein beta-1 (HSPB1) is one of the targets of luteolin action. Luteolin inhibits ferroptosis by promoting the protein expression of HSPB1/solute carrier family 7 member 11 (SLC7A11)/ glutathione peroxidase 4 (GPX4). In conclusion, we have preliminarily elucidated the antioxidant damage ferroptosis ability and the target of action of luteolin to provide a theoretical basis for the application of luteolin in radiation injury diseases.

Fig 1. Protective effects of luteolin on radiation vascular injury.

A. C57BL/6 mice were shaved on the upper back and locally irradiated with 20 Gy of ⁶⁰Co γ-rays, and pre-treated mice were given luteolin by gavage (10 mg/kg, administered by gavage 24 hours before irradiation and 2 hours prior to irradiation for a total of 2 doses). B. Changes in body weight of mice after irradiation with 20 Gy ⁶⁰Coγ rays. Representative graphs of vascular tissue 3 days after irradiation with or without Luteolin (C), H&E staining (E), and Masson trichrome staining (F). Scale bar = 250 μm, cropped image scale bar = 50 μm. #vs control, *vs Ionizing radiation (IR). (#p<0.05, ##p<0.01, *p<0.05, **p<0.01).

Fig 2. Luteolin has anti-ferroptosis ability and low toxicity.

A. Cytotoxicity of human umbilical vein endothelial cells (HUVEC) after treatment with different concentrations of luteolin was detected by Cell Counting Kit 8 (CCK8) assay, n = 5, * vs control. B. The effect of cell viability of HUVEC after treatment with different concentrations of Erastin was detected using the CCK8 assay, n = 5, * vs control. C. Effect of Luteolin and Ferrostatin-1 (fer-1) on cell survival in Erastin-induced ferroptosis in HUVEC by CCK8 assay, n = 5, * vs Erastin. D. Cellular morphological changes were observed using the Giemsa staining method, scale bar = 50 μm. (**p<0.01, ***p<0.001, ### p<0.001).

Fig 3. Luteolin can resist oxidative damage.

A. MDA detection of lipid oxidation after luteolin rescue of HUVECs undergoing ferroptosis, n = 3. B. Changes in SOD enzyme activity after luteolin rescue of HUVECs from ferroptosis, n = 3. C. NADP⁺/NADPH ratio detects changes in intracellular redox state following luteolin rescue of HUVECs from ferroptosis, n = 3. D. Fe²⁺ staining to detect intracellular divalent iron accumulation after luteolin rescue of HUVEC from ferroptosis. E. DHE assay of luteolin rescues the level of oxidative stress in cells after ferroptosis in HUVECs. Scale bar = 100 μm. F. MitoTracker to observe the morphology of mitochondria after luteolin rescue of HUVECs from ferroptosis. Scale bar = 10 μm. #vs control, *vs Erastin. (##p<0.01, ###p<0.001, **p<0.01, ***p<0.001).

Fig 4. Drug affinity responsive target stability (DARTS) identification of target acting proteins of luteolin.

A. Schematic representation of the DARTS process, with a total of 5874 luteolin potential target proteins identified. B-E. Protein enrichment terms after GO, and KEGG pathway analysis are shown on DAVID.

Fig 5. HSPB1 is one of the target proteins of luteolin.

A. Schematic illustration of how computer simulated docking luteolin binds to HSPB1. B. Fitted curves of the interaction results of luteolin with HSPB1 detected by SPR technique. C. Western Blot validation of DARTS results, right panel shows grey value analysis, *vs control. D. Western Blot assay for luteolin affinity for HSPB1, grey value analysis on the right, *vs control. E. Western Blot detection of luteolin-regulated ferroptosis-associated proteins. #vs control, *vs Erastin. (##p<0.01, ###p<0.001, **p<0.01, ***p<0.001).